The sensory hair cells in vestibular organs provide information about head movements to reflexes that control eye, head and body position. Damage to these cells as a result of age, disease or trauma leads to impaired mobility and quality of life. This proposal focuses on the contributions of various classes of ion channel protein to the normal function of hair cells in mammalian vestibular organs. Each of three specific aims is directed at a different stage in stimulus processing: mechanoelectrical transduction, shaping of the receptor potentia1 by voltage-gated ion channels, and afferent transmission. Results will be examined for variation with hair cell type (I vs. II), hair cell location within the sensory epithelium, and developmental stage. Mechanoelectrical transduction is the process by which hair bundle deflection gates mechanosensitive ion channels, producing a transduction current. It has been measured under in vitro conditions that are likely to have changed its properties. One series of experiments will make recording conditions more physiological. The influence of hair bundle morphology on transduction and bundle stiffness is poorly understood and will be examined. The transduction current initiates a voltage change (receptor potential), which activates voltage-gated ion channels. Potassium (K+) channels help set resting potential and provide negative feedback on receptor potentials. The complement of K+ channels in a hair cell varies with cell type and location in the sensory epithelium. K+ channel subunits expressed by single hair cells will be identified by expression profiling: RNA amplified from cDNA from single cells is used to probe a panel of candidate cDNAs. Candidate subunits will be followed up with immunocytochemistry to localize the protein. The voltage-gated sodium conductance (gNa) in mammalian vestibular hair cells inactivates at very negative potentials, raising questions about its function. Mechanisrns controlling the inactivahon range, the effect of gNa on the receptor potential, and the identity of the gNa protein will be investigated. Afferent neurons make large cup-shaped (calyx) endings on type I hair cells, in contrast to the small (bouton) endings they form on other hair cells. Afferent transmission from type I hair cells is therefore likely to have unusual properties. One set of experiments will describe synaptic vesicle exocytosis: Is it different in type I hair cells relative to type II hair cells? Do its properties in mammalian vestibular hair cells differ from those in cochlear hair cells? Another set will test whether the type-I-specific conductance, gK,L, is inhibited by the afferent transmitter present in the synaptic cleft. Such inhibition would produce strong positive feedback on afferent transmission. Ion channels in the postsynaptic calyx ending, which initiate the afferent response to the hair cell transmitter(s), will be characterized.